鲢抗菌肽Hepcidin的基因克隆和表达及抑菌活性分析

利用同源克隆的方法从鲢（Hypophthalmichthys molitrix）的肝脏中克隆出抗菌肽Hepcidin cDNA（GenBank登入号KF312213）后，通过pET32a（+）构建含抗菌肽Hepcidin的重组质粒的pET-Hep/Rosetta菌株，在37 ℃、28 ℃和16 ℃下分别用0.5 mmolL-1和1.0 mmolL-1 IPTG诱导表达，产物用Ni SepharoseTM亲和层析柱纯化并进行体外抑菌试验。鲢Hepcidin cDNA总长度755 bp，ORF为282 bp，5'UTR为108 bp，3'UTR为365 bp，编码93氨基酸，信号肽24个氨基酸，前域42个氨基酸和成熟肽27个氨基酸。pET-Hep/Rosetta在28 ℃和16 ℃主要是可溶性表达，37 ℃主要是包涵体表达。纯化的产物经15% SDS-PAGE电泳验证为目的产物。目的产物、pET32/Rosetta产物以及氟苯尼考的体外抑菌试验表明，目的表达产物对无乳链球菌（Streptococcus agalactiae）、金黄色葡萄球菌（Staphylococcus aureus）、嗜水气单胞菌（Aeromonas hydrophila)等具有很好的抑菌效果。

Cloning,expression and antimicrobial activity of Hepcidin from Hypophthalmichthys molitrix

The full-length Hepcidin cDNA sequence GenBank No. KF312213was amplified from the liver of chub (Hypophthalmichthys molitrix) by semi-nested PCR [rapid amplification of cDNA ends (RACE)].According to prodomain and mature peptide of the Hepcidin cDNA sequence, we designed an upstream primer with EcoR I restriction site and downstream primers with Sal I restriction site, and cloned the target gene into the expression vector pET-32a (+). The recombined plasmid was transformed into the expression stain-E.coli Rosetta and expressed induciblely at different temperatures (37 ℃, 28 ℃ and 16 ℃) and of different IPTG concentrations (0.5 mmolL-1 and 1.0 mmolL-1). The protein was purified by Ni SepharoseTM affinity chromatography column. The full-length of the Hepcidin cDNAgene was 755 bp, which included a 282-bp ORF encoding a 93-amino acid prepropeptide. The prepropeptide contained a signal peptide (24 amino acids), a prodomain (42 amino acids) and a mature peptide (27 amino acids). The purified product displayed a single protein band through 15% SDS-PAGE electrophoresis. The purified production of pET-Hep/Rosetta had obvious antibacterial effect on Streptococcus agalactiae, Staphylococcus aureus andAeromonas hydrophila, but the control group showed no inhibitory effect.